Wiskott-Aldrich syndrome is an X-linked immune deficiency of broad clinical severity arising from mutations in the WASP gene, which results in either a total loss of expression of WAS protein (WASp) or expression of a mutated WASp. WASp, a member of the EVH-1 domain family of proteins, is expressed exclusively in the cells of the hematopoeitic system, and is a known regulator of cellular actin polymerization. The relationship of WASp's effect on actin cytoskeleton and Th dysfunction, however, remains unclear, as in some instances lymphocyte defects in WAS occur despite normal actin polymerization. Our data leads us to speculate on a novel role for WASp - as an adaptor protein bringing together T helper-1 cell-specific signaling molecules and partitioning them to the nucleus where they become part of a Th1-specific transcription complex. Normal activation of the classical Th1 signaling pathway occurs during the Th:DC interaction, resulting in downstream phosphorylation of STAT1 and T-bet, and consequent transcription of IFN-?. In parallel, ITK-dependent phosphorylation of T-bet is required for deactivation of GATA-3, the Th2 transcription factor. A seemingly disparate activation of the Notch signaling pathway also is important for both Th1 and Th2 differentiation. In this proposal, we will test the hypothesis that WASp is a nuclear adaptor protein critical for the correct assembly of a Th1-specific transcriptional complex comprised of members of both the TCR- and Notch-dependent pathways of Th1 activation (NICD, STAT1, and T-bet), and orchestration of Th1 differentiation. We further posit that the severity of WAS clinical phenotype mirrors the degree of Th dysfunction imparted by WASp mutations that disrupt these interactions. In Aim 1, we will test whether WASp is required in the formation of the nuclear macromolecular transcription complex, containing T-bet, NICD, STAT1, and RNA Pol II, and effects transcription of Tbx21 gene under Th1-polarizing condition. In Aim 2, we will test whether the disease-causing WASp missense mutations perturb formation and function of this Th1-nuclear transcription complex. Finally, in Aim 3 we will determine the T cell- specific genes to which WASp binds using genome-wide mapping of in vivo WASp-DNA interactions in the T-cell. These studies should provide new insights into WASp function in the generation of adaptive immunity, and may reveal new therapeutic targets in WAS and other immune deficiencies. PUBLIC HEALTH RELEVANCE: Wiskott-Aldrich syndrome (WAS) is a genetically transmitted systemic immune deficiency resulting from a defect in a protein called WASP and manifesting clinically in severe recurrent infections, easy bruising and bleeding, autoimmunity, and cancers. The studies in this proposal will examine the function of WASP at the molecular level to determine the causes of the above clinical complications of WAS. These studies should lay the foundation for designing novel therapies in the future, for this life-threatening, debilitating disease of the childhood.